This invention relates to a photometric apparatus useful in liquid chromatography (LC) or capillary electrophoresis (CE), and more particularly to such an apparatus which allows efficient fluorescence measurement of small samples while preventing excitation beam light rays from impinging on the cell walls, or unwanted fluorescent signals from affecting the measurement.
In an LC or CE system, detection of sample constituents can be effected by passing the eluent from the column through a small volume sample cell, and passing excitation light through the cell to cause the sample to fluoresce. The fluorescent light emitted, from the cell at right angles to the excitation beam, is measured to determine the characteristics of the sample.
In practice, fluorescence detection from a small volume of sample is limited by the presence of the walls and windows of the sample cell. The walls and windows scatter light at various wavelengths:
1. At the wavelength of the exciting light (Raleigh scattering).
2. At Raman shifted wavelengths characteristic of the cell material.
3. Emission over wide and unpredictable wavelength ranges due to adsorption onto, and penetration into, the cell walls and windows by contaminating fluorescent species. All these contribute to unwanted stray light which cannot be satisfactorily rejected by the emission monochromator or spectrograph. The result is an elevated base line, and noise on the photodetector of photomultiplier signal, raising the minimum detection limit of the species of interest.
In conventional fluorescence systems with small sample volumes, unwanted stray light is minimized by focussing the excitation beam in the center of the sample cell and using masks to collect fluorescent emission only from the center of the cell. However, this means that only a part of the sample can be analyzed, leading to elevated limits of detection.
It has been proposed in U.S. Pat. No. 3,788,744 to provide an apparatus for optically analyzing small particles in a thin liquid stream. The thin stream is surrounded by a flowing liquid sheath in order to prevent contact of the thin stream with the walls of the cell through which it is passed. The particles are caused to fluoresce by an excitation laser beam which is directed transverse the direction of stream flow and the fluorescence is detected at right angles to both. Since the light beam is transverse the direction of the stream flow, the beam traverses the sheath liquid and strikes the walls of the stream conduit thereby causing the problems set forth above.
It has also been proposed in U.S. Pat. No. 3,984,307 to sort particles in a thin stream surrounded by a sheath stream by means of an excition light which permits characterizing the particles to be sorted. However, the excitation light beam also is positioned transverse the thin stream with the attendant problems disclosed above.
It has also been proposed by Fujiwara et al, Anal. Chem. Volume 60, page 1065, 1988 to utilize a high refractive index solvent such as carbon disulfide which is passed through a silica capillary tube but without a sheath stream. A laser light is guided axially by the high refractive index liquid and fluorescent light emerges at the end of the tube along with residual laser light to be filtered and detected. This arrangement does not eliminate the problems associated with light striking the cell wall nor does it utilize total internal reflection to separate the excitation light from the fluorescent emission. Additionally, the requirement of a mobile phase with a refractive index higher than the silica capillary places a severe limitation on possible applications.
Accordingly, it would be desirable to provide a fluorometer capable of detecting induced fluorescence of a sample wherein neither the sample nor excitation light beam contacts the walls or window of a sample cell, and in which full sample volume is measured to achieve maximum sensitivity.